An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells

Rai, Rajeev; Naseem, Asma; Vetharoy, Winston; Steinberg, Zohar; Thrasher, Adrian J.; Santilli, Giorgia; Cavazza, Alessia

doi:10.1016/j.omtm.2023.02.014

Cited by 5 publications

(8 citation statements)

References 56 publications

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“…2b). Unless noted differently, the experiments with undifferentiated CD34 + HSPCs spanned seven days to avoid impairing the stemness features that occur following prolonged cell culture 47 . The GFP + percentages ranged from 0.12% ± 0.05 GFP + cells (mean ± s.d.…”

Section: Resultsmentioning

confidence: 99%

“…Contrary to the large number of chimeric antigen receptor (CAR) T-cells reportedly required in cancer clinical trials (up to 1.0×10 8 cells/kg) 4,21,54 , expanding human CD34 + HSPCs by prolonged cell culture (>7 days) has been reported to detrimentally affect the long-term progenitor cells repopulating capacity 47 . Recent studies of long-term HSPCs transplanted in patients with Wiskott-Aldrich syndrome or β-hemoglobinopathies estimated that as few as 98 edited HSPCs per 10 6 infused CD34 + cells correspond to engrafting cells were sufficient to reconstitute gene-corrected granulocytes in circulation 48 .…”

Section: Discussionmentioning

confidence: 99%

“…Unless otherwise mentioned, the experiments with undifferentiated CD34+ HSPCs spanned seven days given that prolonged cultures impairs the stemness features of the cells 46 . The GFP+ percentages ranged from 0.12% ± 0.05 GFP+ cells (mean ± s.d.…”

Section: Editing Of Gsh Candidates In Human Cd34+ Hspcs With Crispr/c...mentioning

confidence: 99%

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Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Quezada-Ramírez,

Loncar,

Campbell

et al. 2023

Preprint

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Gene transfer into hematopoietic stem and progenitor cells (HSPCs) involving indiscriminately integrating viral vectors has unpredictable outcomes including potential adverse events such as leukemogenesis, resulting from insertional mutagenesis. Therefore, identifying and characterizing genomic safe harbor (GSH) sites where exogenous genetic information can be integrated safely into adult progenitor and stem cells is critically important for therapeutic gene addition. Here, we present a novel approach to identify new GSH sites based on a proven system of stable transgene insertion: the evolutionarily conserved integration of parvovirus DNA into the germlines of host species. From a dataset of 199 unique endogenous parvovirus element (EPV) integration events identified in host genomes, 102 loci were mapped to the human genome with 17 being evaluated for potential use as GSHs in primary human CD34+ HSPCs. Six of the edited loci resulted in sustained transgene expression in both erythroid and myeloid phenotypes while three exhibited myeloid specific-expression and the remaining four loci resulted in a subset of myeloid and erythroid lineages. Following this approach, a minimum of nine promising GSH sites suitable for gene therapy of blood disorders were identified and additional GSH sites are likely to emerge from the remaining mapped loci which may be suitable for gene addition in stem and progenitor cells other than hematopoietic tissues. This novel approach extends the options for gene knock-ins while reducing the risks of insertional mutagenesis, unpredictable expression profiles, and increasing the safety and therapeutic effects.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Quezada-Ramírez,

Loncar,

Campbell

et al. 2023

Preprint

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“…Robust protocols have been developed for CRISPR/Cas9 gene-editing in HSPCs [19,20,21] and several gene editing platforms to treat primary immunodeficiency diseases relying on highfidelity HDR to integrate a therapeutic transgene in its own locus have been developed…”

Section: Introductionmentioning

confidence: 99%

“…This allows targeted therapeutic transgene integration as a platform for correcting multiple mutations [18]. Robust protocols have been developed for CRISPR/Cas9 gene-editing in HSPCs [19,20,21] and several gene editing platforms to treat primary immunodeficiency diseases relying on high-fidelity HDR to integrate a therapeutic transgene in its own locus have been developed preclinically (reviewed in [22]), reaching up to 70% of HSPCs correction and complete haematopoietic reconstitution in mice, establishing the safety and efficacy of this approach.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-based disease modelling and functional correction of Interleukin 7 Receptor alpha Severe Combined Immunodeficiency in T-lymphocytes and hematopoietic stem cells

Rai,

Steinberg,

Romito

et al. 2023

Preprint

Self Cite

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Interleukin 7 Receptor α Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in theIL7RAgene. Defective IL7R expression in humans hampers T cell precursors proliferation and differentiation during lymphopoiesis resulting in absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukaemia, suggesting the application of targeted gene editing to insert a correct copy of theIL7RAgene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modelling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knock-in strategy that targetsIL7RAexon 1 and introduces via homology directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signalling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

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What a Clinician Needs to Know About Genome Editing: Status and Opportunities for Inborn Errors of Immunity

Mudde,

Kuo,

Kohn

et al. 2024

The Journal of Allergy and Clinical Immunology: In Practice

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An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells

Cited by 5 publications

References 56 publications

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

Development of evolutionarily conserved viral integration sites as safe harbors for human gene therapy

CRISPR/Cas9-based disease modelling and functional correction of Interleukin 7 Receptor alpha Severe Combined Immunodeficiency in T-lymphocytes and hematopoietic stem cells

What a Clinician Needs to Know About Genome Editing: Status and Opportunities for Inborn Errors of Immunity

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